Glaucoma remains a major blinding disease affecting over 67 million persons worldwide. An obstruction to aqueous humor outflow, primarily through the trabecular meshwork (TM), produces elevated intraocular pressure (IOP), which is the primary risk factor for glaucomatous optic nerve damage. The exact site and nature of the normal resistance to outflow is poorly defined; the glaucomatous resistance is even more obscure. The resistance appears to reside within the cribriform or juxtacanalicular region of the TM (JCT) and/or Schlemm's canal (SC) inner wall endothelium and to be comprised, at least partially, of extracellular matrix (ECM). Although glaucoma is relatively common, most people do not develop glaucoma, even at advanced age. We, and others, have identified a homeostatic mechanism whereby TM cells sense IOP elevations as mechanical stretching and adjust the outflow resistance to restore normal IOP. This resistance adjustment appears to include ECM turnover. Our immediate goal is to understand the outflow resistance and the molecular and cellular mechanisms by which it is adjusted during this homeostatic response. Our working hypothesis is that manipulations, including the pressure/stretch triggered homeostatic response, which modify the outflow resistance, will produce identifiable changes in the components that form the resistance. Thus, detailed molecular and cellular analysis of this process should enable us to better understand the resistance and its modulation. To this end, we propose two general approaches, 1) to study the actual cellular and molecular processes whereby ECM turnover adjusts the outflow resistance in cell and perfused anterior segment organ culture models; and 2) to evaluate the roles and functions of selected ECM components, which are modulated coincident with manipulations, that change the outflow resistance. These two aims are focused on: 1) podosome/invadopodia-like structures (PILS), which we have just identified on TM cells and in situ in the outflow pathway, that appear to provide controlled and focal EMC turnover and 2) a few matricellular and multidomain ECM organizing proteins that are changed coincident with several outflow resistance manipulations, including pressure/mechanical stretch, TNF1, IL-11 and TGF22. Our preliminary studies strongly support a new paradigm to explain the focused and highly-controlled adjustment of the outflow resistance and its actual physical nature. The proposed studies will provide new insights into the nature of the outflow resistance and its regulation. PUBLIC HEALTH RELEVANCE: Glaucoma remains the second leading cause of vision loss in developed nations. The cause(s) of this disease are not well understood and therapies are directed at treating the symptoms. These studies are focused at understanding what causes glaucoma and why many people do not develop this disease, even at advanced ages.